A major long term objective of this program is the elucidation of the mechanisms involved in the oxidative and peroxidative reactions catalyzed by b-type hemoproteins. The b-type hemoprotein families contain the same iron protoporphyrin prosthetic group, but catalyze quite distinct and different reactions. However, chloroperoxidase occupies a unique niche among the b-type hemoproteins. In terms of catalysis, chloroperoxidase is related to the P-450 cytochromes, the classical plant peroxidases and the catalase family of enzymes. Although chloroperoxidase related to all three of these families in terms of catalytic activity the other three are essentially unrelated to each other. Thus the active site of chloroperoxidase offers an ideal model for understanding the diverse oxidative reactions associated with these hemoproteins. Specific aims include; 1) the elucidation of the three dimensional structure of the active site of chloroperoxidase, 2) the chemical characterization of the electrophilic halogenating intermediate formed in enzymatic halogenation reactions, and 3) the development of chloroperoxidase as a catalyst for the synthesis of chiral epoxides and chiral halogenated products via site directed mutagenesis. An understanding of b-type hemoprotein catalysis is important to the health sciences since many of these enzymes perform vital cellular functions. Steroid hormone synthesis, biosynthesis of prostaglandins and thrombaxanes, detoxification of xenobiotics, and crosslinking reactions after egg fertilization are a few examples of processes which require cytochrome P-450 or peroxidase catalysis. Other important roles for these enzymes involved antibiotic synthesis, lignin degradation and detoxification of environmental pollutants. A second goal of this research is the understanding of the mechanism of activation of pyruvate oxidase by lipid amphiphiles and by limited proteolysis. The catalytic efficiency (k-cat/K-m) of E. coli pyruvate oxidase is increased 450-fold by binding to natural lipid membranes, synthetic lipid vesicles, or a variety of lipid amphiphiles. The amphiphilic activation can be duplicated by cleavage of a 23 amino acid residue peptide from the carboxyl terminal domain of the enzyme. Specific aims of this research involve 1) the identification of the conformational change induced by activation of the enzyme, 2) crystallization of the activated and inactivated forms of the enzyme and 3) determination of the stereochemical course of the reaction.